RESUMO
C-Acylations of polymer-supported 2-phosphoranylidene acetates ("linker reagents") with protected amino acids yielded 2-acyl-2-phosphoranylidene acetates as flexible intermediates for the C-terminal variation of carboxylic acids: peptidyl-2,3-diketoesters, peptidyl vinyl ketones, peptidyl-2-ketoaldehydes, and 1,3-diamino-2-hydroxy-propanes were obtained as products. [reaction: see text]
Assuntos
Acetatos/química , Ácidos Carboxílicos/química , Peptídeos/síntese química , Fosforanos/química , Polímeros/química , Acilação , Aldeídos/química , Técnicas de Química Combinatória , Reagentes de Ligações Cruzadas/química , Diaminas/química , Ésteres/química , Modelos Químicos , Resinas Sintéticas/química , Compostos de Vinila/químicaRESUMO
A synthetic concept is presented that allows the construction of peptide isostere libraries through polymer-supported C-acylation reactions. A phosphorane linker reagent is used as a carbanion equivalent; by employing MSNT as a coupling reagent, the C-acylation can be conducted without racemization. Diastereoselective reduction was effected with L-selectride. The reagent linker allows the preparation of a norstatine library with full variation of the isosteric positions including the P1 side chain that addresses the protease S1 pocket. Therefore, the concept was employed to investigate the P1 site specificity of peptide isostere inhibitors systematically. The S1 pocket of several aspartic proteases including plasmepsin II and cathepsin D was modeled and docked with approximately 500 amino acid side chains. Inspired by this virtual screen, a P1 site mutation library was designed, synthesized, and screened against three aspartic proteases (plasmepsin II, HIV protease, and cathepsin D). The potency of norstatine inhibitors was found to depend strongly on the P1 substituent. Large, hydrophobic residues such as biphenyl, 4-bromophenyl, and 4-nitrophenyl enhanced the inhibitory activity (IC50) by up to 70-fold against plasmepsin II. In addition, P1 variation introduced significant selectivity, as up to 9-fold greater activity was found against plasmepsin II relative to human cathepsin D. The active P1 site residues did not fit into the crystal structure; however, molecular dynamics simulation suggested a possible alternative binding mode.
Assuntos
Indicadores e Reagentes/química , Mimetismo Molecular , Inibidores de Proteases/química , Aminocaproatos/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Catepsina D/antagonistas & inibidores , Protease de HIV/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas de Protozoários , Espectrometria de Massas por Ionização por Electrospray , EstereoisomerismoRESUMO
A method for the parallel solid-phase synthesis of peptide aldehydes has been developed. Protected amino acid aldehydes obtained by the racemization-free oxidation of amino alcohols with Dess-Martin periodinane were immobilized on threonyl resins as oxazolidines. Following Boc protection of the ring nitrogen to yield the N-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin. A peptide aldehyde library was designed for targeting the SARS coronavirus main protease, SARS-CoV M(pro)(also known as 3CL(pro)), on the basis of three different reported binding modes and supported by virtual screening. A set of 25 peptide aldehydes was prepared by this method and investigated in inhibition assays against SARS-CoV M(pro). Several potent inhibitors were found with IC(50) values in the low micromolar range. An IC(50) of 7.5 muM was found for AcNSTSQ-H and AcESTLQ-H. Interestingly, the most potent inhibitors seem to bind to SARS-CoV M(pro) in a noncanonical binding mode.
Assuntos
Aldeídos/síntese química , Biblioteca de Peptídeos , Inibidores de Proteases/síntese química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Aldeídos/isolamento & purificação , Humanos , Espectroscopia de Ressonância Magnética/métodos , Técnicas de Sonda Molecular , Inibidores de Proteases/isolamento & purificaçãoRESUMO
Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.
Assuntos
Isótopos/química , Ligantes , Espectrometria de Massas/métodos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , DNA Complementar/metabolismo , Guanidina/química , Humanos , Complexo Principal de Histocompatibilidade , Nicotina/química , Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray , TransfecçãoRESUMO
Polymer-supported oxidation of alcohols was conducted very efficiently by employing oxoammonium salts, the reactive intermediates in TEMPO oxidations (TEMPO=2,2,6,6-tetramethylpiperidinoxyl). These highly reactive salts (see scheme; X=Br, Cl) could be prepared and isolated on the polymeric support, and were used for the conversion of single compounds as well as of complex mixtures of alcohols.